Rice CYP734As function as multisubstrate and multifunctional enzymes in brassinosteroid catabolism

Catabolism of brassinosteroids regulates the endogenous level of bioactive brassinosteroids. In Arabidopsis thaliana, bioactive brassinosteroids such as castasterone (CS) and brassinolide (BL) are inactivated mainly by two cytochrome P450 monooxygenases, CYP734A1/BAS1 and CYP72C1/SOB7/CHI2/SHK1; CYP734A1/BAS1 inactivates CS and BL by means of C-26 hydroxylation. Here, we characterized CYP734A orthologs from Oryza sativa (rice). Overexpression of rice CYP734As in transgenic rice gave typical brassinosteroid-deficient phenotypes. These transformants were deficient in both the bioactive CS and its precursors downstream of the C-22 hydroxylation step. Consistent with this result, recombinant rice CYP734As utilized a range of C-22 hydroxylated brassinosteroid intermediates as substrates. In addition, rice CYP734As can catalyze hydroxylation and the second and third oxidations to produce aldehyde and carboxylate groups at C-26 in vitro. These results indicate that rice CYP734As are multifunctional, multisubstrate enzymes that control the endogenous bioactive brassinosteroid content both by direct inactivation of CS and by the suppression of CS biosynthesis by decreasing the levels of brassinosteroid precursors.     

Arabidopsis CYP72C1 is an atypical cytochrome P450 that inactivates brassinosteroids

Cytochrome P450 monooxygenases (P450s) are a diverse family of proteins that have specialized roles in secondary metabolism and in normal cell development. Two P450s in particular, CYP734A1 and CYP72C1, have been identified as brassinosteroid-inactivating enzymes important for steroid-mediated signal transduction in Arabidopsis thaliana. Genetic analyses have demonstrated that these P450s modulate growth throughout plant development. While members of the CYP734A subfamily inactivate brassinosteroids through C-26 hydroxylation, the biochemical activity of CYP72C1 is unknown. Because CYP734A1 and CYP72C1 in Arabidopsis diverge more than brassinosteroid inactivating P450s in other plants, this study examines the structure and biochemistry of each enzyme. Three-dimensional models were generated to examine the substrate binding site structures and determine how they might affect the function of each P450. These models have indicated that the active site of CYP72C1 does not contain several conserved amino acids typically needed for substrate hydroxylation. Heterologous expression of these P450s followed by substrate binding analyses have indicated that CYP734A1 binds active brassinosteroids, brassinolide and castasterone, as well as their upstream precursors whereas CYP72C1 binds precursors more effectively. Seedling growth assays have demonstrated that the genetic state of CYP734A1, but not CYP72C1, affected responsiveness to high levels of exogenous brassinolide supporting our observations that CYP72C1 acts on brassinolide precursors. Although there may be some overlap in their physiological function, the distinct biochemical functions of these proteins in Arabidopsis has significant potential to fine-tune the levels of different brassinosteroid hormones throughout plant growth and development.     

BAS1 and SOB7 act redundantly to modulate Arabidopsis photomorphogenesis via unique brassinosteroid inactivation mechanisms

Active brassinosteroids (BRs), such as brassinolide (BL) and castasterone (CS), are growth-promoting plant hormones. An Arabidopsis cytochrome P450 monooxygenase (CYP734A1, formerly CYP72B1), encoded by the BAS1 gene, inactivates BRs and modulates photomorphogenesis. BAS1 was identified as the overexpressed gene responsible for a dominant, BR-deficient mutant, bas1-D. This mutant was isolated in an activation-tagged screen designed to identify redundant genes that might not be identified in classic loss-of-function screens. Here we report the isolation of a second activation-tagged mutant with a BR-deficient phenotype. The mutant phenotype is caused by the overexpression of SOB7 (CYP72C1), a homolog of BAS1. We generated single and double null-mutants of BAS1 and SOB7 to test the hypothesis that these two genes act redundantly to modulate photomorphogenesis. BAS1 and SOB7 act redundantly with respect to light promotion of cotyledon expansion, repression of hypocotyl elongation and flowering time in addition to other phenotypes not regulated by light. We also provide biochemical evidence to suggest that BAS1 and SOB7 act redundantly to reduce the level of active BRs, but have unique mechanisms. Overexpression of SOB7 results in a dramatic reduction in endogenous CS levels, and although single null-mutants of BAS1 and SOB7 have the same level of CS as the wild type, the double null-mutant has twice the amount. Application of BL to overexpression lines of BAS1 or SOB7 results in enhanced metabolism of BL, though only BAS1 overexpression lines confer enhanced conversion to 26-OHBL, suggesting that SOB7 and BAS1 convert BL and CS into unique products.     

Arabidopsis CYP85A2 catalyzes lactonization reactions in the biosynthesis of 2-deoxy-7-oxalactone brassinosteroids

Brassinolide (BL), a plant 7-oxalactone-type steroid hormone, is one of the active brassinosteroids (BRs) that regulates plant growth and development. BL is biosynthesized from castasterone by the cytochrome P450 monooxygenase, CYP85A2. We showed that a Pichia pastoris transformant that synchronously expresses Arabidopsis P450 reductase gene ATR1 and P450 gene CYP85A2 converts teasterone and typhasterol to 7-oxateasterone and 7-oxatyphasterol, respectively. Thus, CYP85A2 catalyzes the lactonization reactions of not only castasterone but also teasterone and typhasterol. The two 2-deoxy-7-oxalactone-type BRs were identified in Arabidopsis plants. Although the reversible conversion between 7-oxateasterone and 7-oxatyphasterol was observed in vivo, no conversion of 7-oxatyphasterol to BL was observed. The biological activity of 7-oxatyphasterol toward Arabidopsis hypocotyl elongation was nearly the same as that of castasterone. These results suggest that a new BR biosynthetic pathway, a BR lactonization pathway, functions in Arabidopsis and plays an important role in regulating the concentration of active BRs, even though the metabolism of 7-oxatyphasterol to BL is still unknown.     

Arabidopsis CYP85A2 Catalyzes Lactonization Reactions in the Biosynthesis of 2-Deoxy-7-oxalactone Brassinosteroids

Brassinolide (BL), a plant 7-oxalactone-type steroid hormone, is one of the active brassinosteroids (BRs) that regulates plant growth and development. BL is biosynthesized from castasterone by the cytochrome P450 monooxygenase, CYP85A2. We showed that a Pichia pastoris transformant that synchronously expresses Arabidopsis P450 reductase gene ATR1 and P450 gene CYP85A2 converts teasterone and typhasterol to 7-oxateasterone and 7-oxatyphasterol, respectively. Thus, CYP85A2 catalyzes the lactonization reactions of not only castasterone but also teasterone and typhasterol. The two 2-deoxy-7-oxalactone-type BRs were identified in Arabidopsis plants. Although the reversible conversion between 7-oxateasterone and 7-oxatyphasterol was observed in vivo, no conversion of 7-oxatyphasterol to BL was observed. The biological activity of 7-oxatyphasterol toward Arabidopsis hypocotyl elongation was nearly the same as that of castasterone. These results suggest that a new BR biosynthetic pathway, a BR lactonization pathway, functions in Arabidopsis and plays an important role in regulating the concentration of active BRs, even though the metabolism of 7-oxatyphasterol to BL is still unknown.     

Tomato cytochrome P450 CYP734A7 functions in brassinosteroid catabolism

Several cytochrome P450 monooxygenases (P450s) catalyze essential oxidative reactions in brassinosteroid (BR) biosynthesis as well as in BR catabolism; however, only limited information exists on the P450s involved in the BR catabolic pathway. Here, we report the characterization of two P450 mRNAs, CYP734A7 and CYP734A8, from Lycopersicon esculentum. These P450s show high homology with Arabidopsis CYP734A1/BAS1 (formerly CYP72B1), which inactivates BRs via C-26 hydroxylation. Transgenic tobacco plants that constitutively overexpressed CYP734A7 showed an extreme dwarf phenotype similar to BR deficiency. Quantitative gas chromatography-mass spectrometry analysis of endogenous BRs in the transgenic plants showed that the levels of castasterone and 6-deoxocastasterone significantly decreased in comparison with those in wild-type plants. By measuring the Type I substrate-binding spectra using recombinant CYP734A7, the dissociation constants for castasterone, brassinolide, and 6-deoxocastasterone were determined to be 6.7, 12, and 12 microM, respectively. In an in vitro assay, CYP734A7 was confirmed to metabolize castasterone to 26-hydroxycastasterone. In addition, 28-norcastasterone and brassinolide were converted to the hydroxylated products. The expression of CYP734A7 and CYP734A8 genes in tomato seedlings was upregulated by exogenous application of bioactive BRs. These results indicated that CYP734A7 is a C-26 hydroxylase of BRs and is likely involved in BR catabolism in tomato. The presence of the CYP734A subfamily in various plant species suggests that oxidative inactivation of BRs by these proteins is a widespread phenomenon in plants.     

Arabidopsis CYP85A2, a cytochrome P450, mediates the Baeyer-Villiger oxidation of castasterone to brassinolide in brassinosteroid biosynthesis

The conversion of castasterone (CS) to brassinolide (BL), a Baeyer-Villiger oxidation, represents the final and rate-limiting step in the biosynthesis of BL in plants. Heterologously expressed Arabidopsis thaliana CYP85A2 in yeast mediated the conversion of CS to BL as well as the C-6 oxidation of brassinosteroids (BRs). This indicated that CYP85A2 is a bifunctional enzyme that possesses BR C-6 oxidase and BL synthase activity. CYP85A2 is thus a cytochrome P450 that mediates Baeyer-Villiger oxidation in plants. Biochemical, physiological, and molecular genetic analyses of Arabidopsis CYP85A2 loss-of-function and overexpression lines demonstrated that CS has to be a bioactive BR that controls the overall growth and development of Arabidopsis plants. Mutant studies also revealed that BL may not always be necessary for normal growth and development but that Arabidopsis plants acquire great benefit in terms of growth and development in the presence of BL.     

The last reaction producing brassinolide is catalyzed by cytochrome P-450s, CYP85A3 in tomato and CYP85A2 in Arabidopsis

Brassinosteroids are steroidal hormones essential for the growth and development of plants. Brassinolide, the most biologically active brassinosteroid, has a seven-membered lactone ring that is formed by a Baeyer-Villiger oxidation of its immediate precursor castasterone. Despite its potential key role in controlling plant development, brassinolide synthase has not been identified. Previous work has shown that the formation of castasterone from 6-deoxocastasterone is catalyzed by members of the CYP85A family of cytochrome P-450 monooxygenases. A null mutation in the tomato Dwarf (CYP85A1) gene, extreme dwarf (d(x)), causes severe dwarfism due to brassinosteroid deficiency, but the d(x) mutant still produces fruits. Here, we show that d(x) fruits contain brassinolide at a higher level than wild-type fruits and that a new CYP85A gene, CYP85A3, is preferentially expressed in tomato fruits. Tomato CYP85A3 catalyzed the Baeyer-Villiger oxidation to produce brassinolide from castasterone in yeast, in addition to the conversion of 6-deoxocastasterone to castasterone. We also show that Arabidopsis CYP85A2, which was initially characterized as castasterone synthase, also has brassinolide synthase activity. Exogenous application of castasterone and brassinolide to the Arabidopsis cyp85a1/cyp85a2 double mutant suggests that castasterone can function as an active brassinosteroid but that its conversion into brassinolide is necessary for normal vegetative development in Arabidopsis. We postulate that castasterone is the major active brassinosteroid during vegetative growth in tomato, whereas brassinolide may play an organ-specific role in fruit development in this species.     

Low-fluence red light increases the transport and biosynthesis of auxin

In plants, light is an important environmental signal that induces photomorphogenesis and interacts with endogenous signals, including hormones. We found that light increased polar auxin transport in dark-grown Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) hypocotyls. In tomato, this increase was induced by low-fluence red or blue light followed by 1 d of darkness. It was reduced in phyA, phyB1, and phyB2 tomato mutants and was reversed by far-red light applied immediately after the red or blue light exposure, suggesting that phytochrome is involved in this response. We further found that the free indole-3-acetic acid (IAA) level in hypocotyl regions below the hook was increased by red light, while the level of conjugated IAA was unchanged. Analysis of IAA synthesized from [13C]indole or [13C]tryptophan (Trp) revealed that both Trp-dependent and Trp-independent IAA biosynthesis were increased by low-fluence red light in the top section (meristem, cotyledons, and hook), and the Trp-independent pathway appears to become the primary route for IAA biosynthesis after red light exposure. IAA biosynthesis in tissues below the top section was not affected by red light, suggesting that the increase of free IAA in this region was due to increased transport of IAA from above. Our study provides a comprehensive view of light effects on the transport and biosynthesis of IAA, showing that red light increases both IAA biosynthesis in the top section and polar auxin transport in hypocotyls, leading to unchanged free IAA levels in the top section and increased free IAA levels in the lower hypocotyl regions.     

Brassinosteroid Mutants Uncover Fine Tuning of Phytochrome Signaling

Phytochromes (phy) A and B provide higher plants the ability to perceive divergent light signals. phyB mediates red/far-red light reversible, low fluence responses (LFR). phyA mediates both very-low-fluence responses (VLFR), which saturate with single or infrequent light pulses of very low fluence, and high irradiance responses (HIR), which require sustained activation with far-red light. We investigated whether VLFR, LFR, and HIR are genetically coregulated. The Arabidopsis enhanced very-low-fluence response1 mutant, obtained in a novel screening under hourly far-red light pulses, showed enhanced VLFR of hypocotyl growth inhibition, cotyledon unfolding, blocking of greening, and anthocyanin synthesis. However, eve1 showed reduced LFR and HIR. eve1 was found allelic to the brassinosteroid biosynthesis mutant dim/dwarf1. The analysis of both the brassinosteroid mutant det2 in the Columbia background (where VLFR are repressed) and the phyA eve1 double mutant indicates that the negative effect of brassinosteroid mutations on LFR requires phyA signaling in the VLFR mode but not the expression of the VLFR. Under sunlight, hypocotyl growth of eve1 showed little difference with the wild type but failed to respond to canopy shadelight. We propose that the opposite regulation of VLFR versus LFR and HIR could be part of a context-dependent mechanism of adjustment of sensitivity to light signals.     

Castasterone Can be Biosynthesized from 28-homodolichosterone in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

We recently demonstrated the biosynthesis of 24-ethylidene brassinosteroids in Arabidopsis thaliana. To determine the physiological role of biosynthesis of 24-ethylidene brassinosteroids, metabolism of 28-homodolichosterone as the end product of 24-ethylidene brassinosteroids biosynthesis was examined by a crude enzyme solution prepared from A. thaliana. In wild-type plants, dolichosterone and castasterone were identified as enzyme products on GC-MS analysis. In a mutant where DWARF1 was overexpressed (35S-DWF1), the conversion rate of 28-homodolichosterone to castasterone was significantly increased. These results indicate that conversion of 28-homodolichosterone to castasterone is mediated by dolichosterone in Arabidopsis. In the root growth assay, inhibitory activity was enhanced in the order of castasterone > dolichosterone > 28-homodolichosterone, demonstrating that conversion of 28-homodolichosterone to castasterone via dolichosterone is a biosynthetic reaction that increases BR activity in Arabidopsis. Compared to Arabidopsis grown under dark conditions, light-grown Arabidopsis showed up-regulated DWARF1 expression, resulting in an increased conversion rate of 28-homodolichosterone to castasterone, suggesting that light is an important regulatory factor for the biosynthetic connection of 24-ethylidene brassinosteroids and 24-methyl brassinosteroids in A. thaliana. Consequently, 24-ethylidene brassinosteroids biosynthesis to generate 28-homodolichosterone is a lightregulated alternative route for synthesis of the biologically-active BRs, castasterone and brassinolide in Arabidopsis plants.     

Accumulation of 6-deoxocathasterone and 6-deoxocastasterone in Arabidopsis, pea and tomato is suggestive of common rate-limiting steps in brassinosteroid biosynthesis

To gain a better understanding of brassinosteroid biosynthesis, the levels of brassinosteroids and sterols related to brassinolide biosynthesis in Arabidopsis, pea, and tomato plants were quantified by gas chromatography-selected ion monitoring. In these plants, the late C-6 oxidation pathway was found to be the predominant pathway in the synthesis of castasterone. Furthermore, all these plant species had similar BR profiles, suggesting the presence of common biosynthetic control mechanisms. The especially high levels of 6-deoxocathasterone and 6-deoxocastasterone may indicate that their respective conversions to 6-deoxoteasterone and castasterone are regulated in planta and hence are important rate-limiting steps in brassinosteroid biosynthesis. Other possible rate-limiting reactions, including the conversion of campestanol to 6-deoxocathasteonre. are also discussed. Tomato differs from Arabidopsis and pea in that tomato contains 28-norcastasterone as a biologically active brassinosteroid, and that its putative precursors, cholesterol and its relatives are the major sterols.     

Genetic Evidence That the Red-Absorbing Form of Phytochrome B Modulates Gravitropism in Arabidopsis thaliana

Hypocotyls of dark-grown Arabidopsis seedlings exhibit strong negative gravitropism, whereas in red light, gravitropism is strongly reduced. Red/far-red light-pulse experiments and analysis of specific phytochrome-deficient mutants indicate that the red-absorbing (Pr) form of phytochrome B regulates normal hypocotyl gravitropism in darkness, and depletion of Pr by photoconversion to the far-red-absorbing form attenuates hypocotyl gravitropism. These studies provide genetic evidence that the Pr form of phytochrome has an active function in plant development.     

Bioactivity of 25-hydroxy-, 26-hydroxy, 25,26-dihydroxy- and 25,26-epoxybrassinolide.

The bioactivity of 25-hydroxybrassinolide, (25S)- and (25R)-26-hydroxybrassinolide, (25S)- and (25R)-25,26-dihydroxybrassinolide, and of (25R)-25,26-epoxybrassinolide was tested in the rice leaf lamina inclination assay. The 25- and (25S)-26-hydroxy derivatives are known metabolites of the naturally-occurring phytohormone brassinolide, whereas the other compounds are novel, but closely related, congeners. When tested alone, all showed either no activity or only weak activity at relatively high doses. When coapplied with indole-3-acetic acid (IAA), an auxin that synergizes the effects of brassinosteroids, enhanced bioactivity was observed for each compound. However, even when applied together with IAA, none of the compounds proved more bioactive than brassinolide with or without IAA. We conclude from these results that enzymatic hydroxylation of endogenous brassinolide at C-25 and/or C-26 does not enhance brassinosteroid activity, and so does not comprise an activation pathway in brassinolide biosynthesis. Instead, these hydroxylations result in modest to appreciable metabolic deactivation.     

A brassinosteroid-insensitive mutant in Arabidopsis thaliana exhibits multiple defects in growth and development.

Brassinosteroids are widely distributed plant compounds that modulate cell elongation and division, but little is known about the mechanism of action of these plant growth regulators. To investigate brassinosteroids as signals influencing plant growth and development, we identified a brassinosteroid-insensitive mutant in Arabidopsis thaliana (L.) Henyh. ecotype Columbia. The mutant, termed bri1, did not respond to brassinosteroids in hypocotyl elongation and primary root inhibition assays, but it did retain sensitivity to auxins, cytokinins, ethylene, abscisic acid, and gibberellins. The bri1 mutant showed multiple deficiencies in developmental pathways that could not be rescued by brassinosteroid treatment including a severely dwarfed stature; dark green, thickened leaves; males sterility; reduced apical dominance; and de-etiolation of dark-grown seedlings. Genetic analysis suggests that the Bri1 phenotype is caused by a recessive mutation in a single gene with pleiotropic effects that maps 1.6 centimorgans from the cleaved, amplified, polymorphic sequence marker DHS1 on the bottom of chromosome IV. The multiple and dramatic effects of mutation of the BRI1 locus on development suggests that the BRI1 gene may play a critical role in brassinosteroid perception or signal transduction.     

Selective interaction of triazole derivatives with DWF4, a cytochrome P450 monooxygenase of the brassinosteroid biosynthetic pathway, correlates with brassinosteroid deficiency in planta

Brassinazole, a synthetic chemical developed in our laboratory, is a triazole-type brassinosteroid biosynthesis inhibitor that induces dwarfism in various plant species. The target sites of brassinazole were investigated by chemical analyses of endogenous brassinosteroids (BRs) in brassinazole-treated Catharanthus roseus cells. The levels of castasterone and brassinolide in brassinazole-treated plant cells were less than 6% of the levels in untreated cells. In contrast, campestanol and 6-oxocampestanol levels were increased, and levels of BR intermediates with hydroxy groups on the side chains were reduced, suggesting that brassinazole treatment reduced BR levels by inhibiting the hydroxylation of the C-22 position. DWF4, which is an Arabidopsis thaliana cytochrome P450 isolated as a putative steroid 22-hydroxylase, was expressed in Escherichia coli, and the binding affinity of brassinazole and its derivatives to the recombinant DWF4 were analyzed. Among several triazole derivatives, brassinazole had both the highest binding affinity to DWF4 and the highest growth inhibitory activity. The binding affinity and the activity for inhibiting hypocotyl growth were well correlated among the derivatives. In brassinazole-treated A. thaliana, the CPD gene involved in BR biosynthesis was induced within 3 h, most likely because of feedback activation caused by the reduced levels of active BRs. These results indicate that brassinazole inhibits the hydroxylation of the C-22 position of the side chain in BRs by direct binding to DWF4 and that DWF4 catalyzes this hydroxylation reaction.     

Genetic analysis of the roles of phytochromes A and B1 in the reversed gravitropic response of the lz-2 tomato mutant

The lz-2 mutation in tomato ( Lycopersicon esculentum ) causes conditional reversal of shoot gravitropism by light. This response is mediated by phytochrome. To further elicit the mechanism by which phytochrome regulates the lz-2 phenotype, phytochrome-deficient lz-2 plants were generated. Introduction of au alleles, which severely block chromophore biosynthesis, eliminated the reversal of hypocotyl gravitropism in continuous red and far-red light. The fri ¹ and tri ¹ alleles were introduced to specifically deplete phytochromes A and B1, respectively. In dark-grown seedlings, phytochrome A was necessary for response to high-irradiance far-red light, a complete response to low fluence red light, and also mediated the effects of blue light in a far-red reversible manner. Loss of phytochrome B1 alone did not significantly affect the behaviour of lz-2 plants under any light treatment tested. However, dark-grown lz-2 plants lacking both phytochrome A and B1 exhibited reduced responses to continuous red and were less responsive to low fluence red light and high fluence blue light than plants that were deficient for phytochrome A alone. In high light, full spectrum greenhouse conditions, lz-2 plants grew downward regardless of the phytochrome deficiency. These results indicate that phytochromes A and B1 play significant roles in mediating the lz-2 phenotype and that at least one additional phytochrome is involved in reversing shoot gravitropism in this mutant.